1. Field of the Invention
The invention relates to an expression system for production of proteins in fungi of the genera Trametes or Polyporus, to its preparation and to its use.
2. The Prior Art
Various prokaryotic and eukaryotic expression systems are known for protein production. Examples of prokaryotic expression systems are Escherichia coli and Bacillus subtilis. The methods for genetic manipulation of these organisms are well established. Specific disadvantages of these expression systems are the frequently disappointingly low production rate in particular of eukaryotic proteins, the folding of the produced proteins in such a way that they are often not in active form, and, in particular, the absence of the post-translational modification of the expressed proteins. As example of the absence of post-translational modification, mention may be made of the absence of incorporation of prosthetic groups or the absence of glycosylation of the protein to be expressed.
These disadvantages of prokaryotic expression systems can be avoided by using eukaryotic systems.
Widespread eukaryotic expression systems which are widely used include cell culture systems both of mammalian cells and insect cells, and eukaryotic microorganisms such as yeasts or filamentous fungi. Whereas the protein to be expressed is usually produced in active form with these expression systems, the production rate is in many cases too low, especially on expression of heterologous proteins. Expression in the yeast Saccharomyces cerevisiae or in filamentous fungi from the ascomycetes class may serve as example thereof.
High production rates in filamentous fungi such as Aspergillus have been described in particular for the expression of homologous proteins or proteins of filamentous fungi of the ascomycetes class. Expression of heterologous proteins often takes place with only low or moderate yields. WO 96/00290, for example, describes heterologous expression of the laccase LCC1 from the filamentous fungus of the basidiomycetes class, Polyporus pinsitus. On expression in Aspergillus, a filamentous fungus of the ascomycetes class, yields of up to 0.35 g/l are obtained in the fermentation. This is a comparatively small increase in yield compared with the production of 0.1-0.2 g/l by a comparable wild-type strain in the fermentation.
There is an increasing interest in industrial applications especially for enzymes from the basidiomycetes class and therein especially the white rot fungi. Examples which may be mentioned are hydrolytic enzymes such as cellulases, hemicellulases or lipases, or else oxidoreductases such as lignin peroxidases, manganese peroxidases, laccases, cellobiose-quinone oxidoreductase or cellobiose oxidase. Potential applications for these enzymes exist, for example, in wood and pulp processing.
One class of enzymes occurring among others in basidiomycetes and of great interest for industrial applications is the class of laccase enzymes (p-hydroxyphenol oxidase, EC 1.10.3.2.). Laccases belong to the protein family called the xe2x80x9cblue copper proteinsxe2x80x9d and usually contain four copper ions which are arranged in three copper centers referred to as type 1 to type 3. Laccases are further distinguished by generally being secreted proteins and possibly containing a glycosylation content of up to 10 to 45% of the molecular weight. Beside the depolymerization of macromolecular compounds such as lignin, laccases are also able to catalyze the polymerization in particular of aromatic compounds. An example thereof is lignin biosynthesis in plants, in which the laccases present in plants are involved. Possible industrial applications of laccases are in paper manufacture for the delignification of pulp, in polymerization reactions of all types, for example in waste water treatment. The use of laccases in organic chemical synthesis is also known, for example in coupling reactions or the side-chain oxidation of aromatic compounds. However, a precondition for industrial application of all these processes is that the laccase enzyme can be provided at reasonable cost and in relatively large amounts.
DNA vectors said to be suitable for transformation and selection of transformants have been described for various filamentous fungi from the basidiomycetes class. A method for homologous transformation of the basidiomycete Phanerochaete chrysosporium has been described (M. Alic et al. (1991) Curr. Genet. 19, 491-494). DNA constructs for transformation of the basidiomycete Pleurotus ostreatus has been described (K. Yanai et al. (1996) Biosci. Biotech. Biochem. 60, 472-475). U.S. Pat No. 5,362,640 describes DNA vectors for the transformation of the basidiomycete Coriolus hirsutus. Likewise, a DNA vector for transformation of the basidiomycete Coriolus versicolor has been described (Y. Iimura et al. (1992) 5th International Conference on Biotechnology in the Pulp and Paper Industry, 427-431). It has not been disclosed for any of these expression systems from the basidiomycetes class that a significant increase in the expression rate for homologous or heterologous proteins has been achieved.
Known expression vectors containing genetic regulatory elements for expression in filamentous fungi of the ascomycetes class cannot be efficiently expressed in filamentous fungi of the basidiomycetes class. Thus, on transformation of filamentous fungi of the basidiomycetes class they do not allow the selection of positive transformants on the basis of, for example, acquired antibiotic resistance or the expression of a color-forming indicator protein or on the basis of the complementation of an auxotrophic gene defect.
The present invention relates to an expression system for the production of a protein in a filamentous fungi consisting of
a) a host organism selected from the genera Trametes and Polyporus and
b) a DNA vector which comprises a selection marker gene which codes for a protein which, after transformation of the host organism, allows selection of positive transformants and is selected from the group of antibiotic resistance genes which code for proteins which abolish the growth-inhibiting effect of antibiotics to which the host organism is not resistant, of genes which encode proteins which are capable of a color-forming reaction, and of genes which complement a genetic defect in the host organism (auxotrophy), where expression of the selection marker gene is controlled by at least one genetic regulatory element which is active in the host organism, and
c) a DNA vector which comprises a gene which codes for the protein to be produced, where expression of this gene and, where appropriate, also secretion of the protein thus produced is controlled by a genetic regulatory element which is active in the host organism, where the DNA vector which comprises a selection marker gene, and the DNA vector which comprises the gene which codes for the protein to be produced may also be present as a DNA vector.
Suitable and preferred as antibiotic resistance genes are genes which confer resistance to an antibiotic from the group of hygromycin, bialaphos, kanamycin, geneticin, bleomycin, oligomycin, G418, zeocin, benomyl and phleomycin.
It is possible and preferred to use further selection marker genes which code for proteins which are capable of a color-forming reaction, for example the glucuronidase gene or the gene for green fluorescent protein (GFP).
Selection marker genes able to complement a genetic defect in the host organism (auxotrophy) are particularly suitable.
Host organisms preferred for the expression system according to the invention are monokaryotic strains from the genera Trametes and Polyporus.
Host organisms of the species Trametes versicolor are particularly preferred.
The host organism in the expression system according to the invention is preferably distinguished by also having a genetic defect in metabolism (auxotrophy), on the basis of which one or more metabolites essential for growth can no longer be synthesized, and the host organism is no longer able to grow on minimal media without addition of this or these metabolites.
The invention therefore also relates to an expression system wherein the host organism selected from the genera Trametes and Polyporus has a genetic defect in metabolism (auxotrophy) on the basis of which one or more metabolites essential for growth can no longer be synthesized, and the host organism is no longer able to grow on minimal media without addition of this or these metabolites, and the selection marker gene is selected in such a way that it complements the auxotrophic gene defect of the host organism.
Examples of selection marker genes able to complement an auxotrophic gene defect in the host organism are the OCT gene (codes for ornithine carbamoyltransferase, U.S. Pat. No. 5,362,640), the pyr G gene (codes for orotidine-5xe2x80x2-phosphate decarboxylase, Goosen et al., Curr. Genet. (1987) 11, 499-503), the trpC gene (codes for a trifunctional gene product which has enzymic activity for phosphoribosylanthranilate isomerase, glutamine amido-transferase and indolglycerol-phosphate synthase, Yelton et al., Proc Natl. Acad. Sci. USA (1984) 81, 1470-1474), or the nia D gene (codes for nitrate reductase).
The particularly preferred selection marker gene is the pyr G gene which codes for orotidine-5xe2x80x2-phosphate decarboxylase (an enzyme of uridine metabolism) and which is able to complement the uridine auxotrophy of a host strain deficient in the pyrG gene. The host organism which is particularly preferred in this case is a strain from the genus Trametes or Polyporus having a defect in the pyr G gene and auxotrophic for uridine.
The pyr G gene is preferably derived from a fungus from the basidiomycetes class, for example from the genus Agaricus, Coriolus, Polyporus, Pleurotus, Phanerochaete, Schizophyllum or Trametes.
Suitable and preferred for expression of the pyr G gene are the promoter and terminator elements for a glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH gene), for example from the filamentous fungi from the basidiomycetes class Schizophyllum commune, Agaricus bisporus (the GAPDH Ag2 gene), Phanerochaete chrysosporium, Trametes versicolor or for a laccase gene, preferably the laccase I gene, or the laccase III gene from Trametes versicolor. 
Particularly suitable as selection marker gene for the expression system according to the invention are the orotidine-5xe2x80x2-phosphate decarboxylase gene (pyr G gene), preferably from the basidiomycete Schizophyllum commune or from the basidiomycete Trametes versicolor. 
Expression of the pyr G gene from the basidiomycete Schizophyllum commune is preferably controlled by the promoter and, where appropriate, terminator of the pyr G gene from Schizophyllum commune. 
Expression of the pyr G gene from the basidiomycete Trametes versicolor is preferably controlled by the promoter of the pyr G gene from Trametes versicolor. 
The expression system according to the invention is particularly suitable for expressing a gene which codes for a hydrolytic enzyme, for example from the group of cellulases, hemicellulases and lipases, or from the group of oxidoreductases such as, for example, the lignin peroxidases, manganese peroxidases, laccases, cellobiose-quinone oxidoreductase or cellobiose oxidase.
It is particularly preferably suitable for expression of a gene for a laccase.
The invention further relates to a DNA vector which comprises at least one selection marker gene which codes for a protein which, after transformation of a fungus selected from the genera Trametes and Polyporus, allows selection of positive transformants, wherein the selection marker gene is selected from the group of antibiotic resistance genes which code for proteins which abolish the growth-inhibiting effect of antibiotics to which the host organism is not resistant, of genes which encode proteins which are capable of a color-forming reaction, and of genes which complement a genetic defect in the host organism (auxotrophy), and wherein the selection marker gene is controlled by at least one genetic regulatory element active in the host organism.
The DNA vectors according to the invention allow the selection of positive transformants on the basis of complementation of an auxotrophic gene defect in the host organism on transformation of fungi selected from the genera Trametes and Polyporus.
In particular, the selection of transformants of the filamentous fungus Trametes versicolor and, in a particularly preferred embodiment, transformants of pyr G-deficient auxotrophic strains of Trametes versicolor is made possible by the genes mentioned as particularly preferred for complementation of auxotrophic gene defects in the host organism.
The DNA vectors according to the invention are also suitable for expression of genes which code for proteins in a host organism of the genus Trametes and Polyporus. Genes which code for proteins mean for the purpose of the invention also the cDNA genes derived from the structural genes for the proteins. The proteins may be proteins which are heterologous for the host organism or proteins which are homologous for the host organism.
The DNA vector according to the invention thus preferably also comprises at least one gene which codes for a protein to be expressed.
The DNA vector according to the invention particularly preferably comprises at least one gene which codes for a hydrolyzing enzyme, for example from the group of cellulases, hemicellulases and lipases, or from the group of oxidoreductases such as, for example, lignin peroxidases, manganese peroxidases, laccases, cellobiose-quinone oxidoreductase or cellobiose oxidase.
The DNA vector according to the invention particularly preferably comprises a gene for a laccase.
A promoter which is necessary for expression of the protein-encoding gene may originate from the gene to be expressed, or it is also possible to use the promoter of a foreign gene functionally linked to the coding region of the gene to be expressed.
The DNA vector according to the invention thus preferably also comprises a promoter for expression of the protein-encoding gene.
The DNA vector according to the invention particularly preferably comprises as promoter for expression of the protein-encoding gene a promoter which ensures a high level of expression.
A promoter preferably used for this purpose is, for example, the promoter of the gene for the protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for example from the species Trametes versicolor. 
The DNA vector according to the invention preferably also comprises a transcription terminator for the protein-encoding gene.
It is possible to use as transcription terminator the terminator of the protein-encoding gene to be expressed or else the terminator of a foreign gene. The transcription terminator of the gene of a laccase is preferred.
Expression of the proteins can take place intracellularly or, in the presence of a signal sequence capable of functioning for the purpose of secretion, also extra-cellularly.
If secretion of the expressed protein from the cell is desired, the DNA vector according to the invention preferably comprises a signal sequence capable of functioning 5xe2x80x2 upstream of the protein-encoding gene. It is additionally possible for a so-called secretion carrier, functionally linked to the 5xe2x80x2 end of the protein-encoding gene, to be present in the DNA vector according to the invention.
The secretion carrier can be the gene for a secreted protein or the fragment of a gene for a secreted protein. The secretion carrier can be functionally linked to the protein to be secreted in such a way that a fusion protein is produced from the secretion carrier and the protein to be secreted. In another embodiment, the linkage of secretion carrier and the protein to be secreted is designed so that the secretion carrier can be separated from the protein to be secreted. This can be brought about, for example, by inserting a recognition sequence for a protein-cleaving enzyme into the linkage site between secretion carrier and the protein to be secreted. An example of this which may be mentioned is the lysine-arginine recognition sequence for the so-called KEX2 protease and an example of a secretion carrier is the glucoamylase from Aspergillus niger (Contreras et al., Bio/Technology (1991) 9, 378-381, Broekhuijsen et al., J. of Biotechnology (1993) 31, 135-145).
DNA sequences which are involved other than as transcription terminators at the 3xe2x80x2 end of the protein-encoding gene in the expression and secretion of the expressed protein can likewise be present in the DNA vector according to the invention. One example thereof is provided by the gene for the laccase from Neurospora crassa, whose 3xe2x80x2 end contains the sequence for 13 amino acids which are deleted during secretion of the protein and are no longer present in the mature protein (Germann et al., J. Biol. Chem. (1988) 263, 885-896).
Preparation of the DNA vectors according to the invention takes place by methods known in the prior art. Various possibilities are explained in the examples. The methods described therein can be applied by the skilled worker to any desired other vectors, resistance genes, regulatory elements and structural genes.
The DNA vectors according to the invention are suitable for producing fungal strains which are capable of efficient expression and secretion of proteins.
The invention therefore also relates to processes for the production of fungal strains which are capable of the efficient expression and secretion of proteins.
This process comprises using as host organism a fungus selected from the genera Trametes and Polyporus which has an auxotrophic gene defect and which is transformed by methods known per se with a DNA vector which has a gene for complementation of the auxotrophic gene defect in the host strain, and selecting from the transformation mixture the clones transformed with the DNA vector by selection for complementation of the auxotrophic gene defect, where expression of the gene for complementation of the auxotrophic gene defect in the host strain is controlled by a genetic regulatory element which is active in the host strain.
Filamentous fungi which can be used as host for the gene expression belong to the genus Trametes or Polyporus.
The preferred host for the gene expression is a mono-karyotic basidiomycete from the genus Trametes or Polyporus.
It is a further advantage for said host strains from the basidiomycetes class to have an auxotrophic gene defect which can be used as selection marker for identifying positive transformants. It is possible to use, for example, host strains from the basidiomycetes class which have a gene defect in the OCT gene, in the pyr G gene, in the trpC gene or in the nia D gene.
The preferred host for gene expression is a fungus selected from the genera Trametes and Polyporus and having a defect in the pyr G gene.
Particularly preferred for gene expression is a host of the species Trametes versicolor which has a defect in the pyr G gene and is auxotrophic for uridine.
Transformation of the host strain takes place by methods corresponding to the prior art. These methods include transformation of protoplasts by the CaCl2/PEG method, transformation by electroporation or biolitic transformation by bombardment with DNA-containing microprojectiles. These methods are described in standard textbooks.
For example, the gene to be transformed is cloned in a known manner into a DNA vector according to the invention and introduced by the methods mentioned into a filamentous fungus selected from the genera Trametes and Polyporus.
However, the gene to be transformed can also be cloned into an expression vector without selection marker gene and used together with a vector which complements the auxotrophic gene defect in the host strain for generating transformants (cotransformation).
The preferred strain for the transformation is a filamentous fungus selected from the genera Trametes and Polyporus. The relevant strain from the basidiomycetes class can moreover be a monokaryotic or else a dikaryotic strain. In a preferred embodiment, it is a uridine-auxotrophic strain which has a defect in the pyr G gene.
Particularly preferred for the transformation is a mono-karyotic, uridine-auxotrophic, pyr G-deficient strain from the species Trametes versicolor. 
The selection of positive transformants takes place, for example, by placing protoplasts, after transformation with vector DNA, on a medium to which is added, for osmotic stabilization of the protoplasts, an addition such as, for example, sorbitol, mannitol or sucrose and which allows the selection of transformants with the complementing pyr G gene.
In a preferred embodiment of the invention, the filamentous fungus Trametes versicolor is transformed in a homologous system with the gene of a laccase from Trametes versicolor. This achieves an increase in the expression rate for said laccase, which significantly improves the production rate of 0.1 g of laccase/l of culture medium in the fermentation which can be achieved in the prior art.
Preferably used for this purpose is the promoter which is intrinsic to the laccase gene or the promoter for a strongly expressed gene from Trametes versicolor. The promoters of the laccase genes I and III, whose isolation is described in the 4th example, are preferably used. This entails using from the laccase I gene preferably the sequence section from base 1-1192 contained in SEQ ID NO: 1. The sequence section from base 1-547 of the laccase III gene contained in SEQ ID NO: 2 is preferably used. The promoter of another strongly expressed gene is represented by the GAPDH promoter for the glyceraldehyde-3-phosphate dehydrogenase from Trametes versicolor. Isolation of the GAPDH promoter is described in the 5th example. The GAPDH promoter sequence corresponds to the sequence section listed in SEQ ID NO: 3, base 1-1542. It has emerged that, in particular, the sequence section in SEQ ID NO: 3, base 1365 to bp 1542, and sequences homologous thereto and having a homology of greater than 73% are suitable for the expression. It is further preferred for at least one of the following sequence sections likewise to be present on the regulatory element according to the invention:
SEQ ID NO: 3: base 1005 to bp 1123, and sequences homologous to this sequence section and having a homology of greater than 52%; or
SEQ ID NO: 3: base 817 to bp 947, and sequences homologous to this sequence section and having a homology of greater than 44%, or
SEQ ID NO: 3: base 640 to bp 728, and sequences homologous to this sequence section and having a homology of greater than 50%.
All homology levels mentioned in the present invention relate to results obtained with the computer program xe2x80x9cWisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, Wis.xe2x80x9d. The homology determination takes place by searching the database with the subprogram xe2x80x9cfastaxe2x80x9d and the preset values (word size 6). The most similar sequences are then examined for homology with the subprogram xe2x80x9cgapxe2x80x9d. The preset parameters xe2x80x9cgap creation penalty 50xe2x80x9d. and xe2x80x9cgap extension penalty 3xe2x80x9d are used for this. In addition, the subprogram xe2x80x9cgapxe2x80x9d and the above-mentioned preset parameters were used to examine for homology the promoter sequence, which is disclosed in JP 09047289 but which is not yet available in a database, of the GAPDH gene from Coriolus hirsutus. 
The invention thus also relates to a regulatory element which is active in Trametes or Polyporus and which comprises the sequence section from base 1-1192 present in SEQ ID NO: 1 or the sequence section from base 1-547 present in SEQ ID NO: 2 or the sequence section from base 1365-1542 present in SEQ ID NO: 3, and sequences homologous to this sequence section and having a homology of greater than 73%.
The present invention also provides a regulatory element which is active in Trametes and Polyporus comprising a sequence section selected from the group consisting of the sequence section from base 1-1192 present in SEQ ID NO: 1, the sequence section from base 1-547 present in SEQ ID NO: 2 and the sequence section from base 1365-1542 present in SEQ ID NO: 3 and regulatory elements derived thereof by base substitutions amounting to a sequence change of no more than 27%.
The selection media preferably used are those on which only Trametes versicolor transformants which have been transformed with a functionally expressed selection marker gene for the pyr G gene are able to grow. Preference is given to the minimal medium described in the 2nd example in the absence of uridine, on which pyr G-auxotrophic strains of Trametes versicolor are no longer able to grow, or can grow further only after addition of uridine.
Successful use of a DNA vector according to the invention containing the pyr G gene as selection system depends on efficient expression of the selection marker gene in Trametes transformants. Appropriate expression signals are necessary for efficient expression.
Expression signals from basidiomycetes bring about functional expression in Trametes versicolor with, surprisingly, considerably greater efficiency than the expression signals otherwise available from ascomycetes. For this reason, the pyr G selection marker gene in the DNA vectors according to the invention is preferably under the control of genetic regulatory elements from basidiomycetes, particularly preferably from those selected from the genera Trametes and Polyporus.
The pyr G gene is preferably under the control of the 5xe2x80x2 promoter region upstream of it, and the 3xe2x80x2 terminator region downstream of it. A DNA fragment in which the pyr G gene from Schizophyllum commune is under the control of the expression signals of the pyr G gene from Schizophyllum commune is described by Froeliger et al., Gene (1989) 83, 387-393. The pyr G gene may also be under the control of expression signals from basidiomycetes which differ from those of the pyr G gene. Expression signals which comply with this function include GAPDH promoters of filamentous fungi from the basidiomycetes class, such as, for example, Coriolus hirsutus, Phanerochaete chrysosporium, Agaricus bisporus or Trametes versicolor, the OCT promoter from Coriolus hirsutus, the promoter of laccase I or laccase III from Trametes versicolor and the terminator of the GAPDH gene from Agaricus bisporus or the terminators of the laccase I or laccase III gene from Trametes versicolor. 
Particular preference is given to a vector which is described in the 3rd example, in which the pyr G gene from Schizophyllum commune is under the control of the expression signals of the pyr G gene from Schizophyllum commune. 
The pyr G gene can be any gene which codes for a protein having the enzymatic activity of an orotidine-5xe2x80x2-phosphate decarboxylase. The pyr G gene preferably originates from a filamentous fungus from the basidiomycetes class, such as, for example, Agaricus bisporus, Phanerochaete chrysosporium, Coriolus hirsutus, Polyporus pinsitus, Schizophyllum commune or Trametes versicolor. 
The pyr G gene from Schizophyllum commune and Trametes versicolor is particularly preferred.
The invention further relates to fungal strains selected from the genera Trametes and Polyporus which comprise a DNA vector according to the invention.
These fungal strains are capable of efficient expression and secretion of proteins.
The invention thus also relates in particular to filamentous fungi of the genus Trametes or Polyporus which comprise a DNA vector according to the invention.
The expressed and secreted proteins may be heterologous proteins or else homologous proteins.
Preference is given to a laccase. Such laccases are known from the strain Trametes versicolor for example (examples of laccase genes are the gene for laccase I: SEQ ID NO: 1, base 1193-3312, or the gene for laccase III: SEQ ID NO: 2, base 548-2542).
The invention thus also relates to a process for production of proteins which comprises employing the expression system according to the invention in a manner known per se for protein production, or comprises cultivating in a manner known per se a fungal strain which has been produced by the process according to the invention.
Such production processes are disclosed, for example, in CA: AN 96-203142, Eggert et al., Appl. Environ. Microbiol (1996) 62, 1151-1158, Martinez et al., Appl. Microbiol. Biotechnol. (1994) 41, 500-504, or WO 93/08272.